JP4620404B2 - Yeast mutant, method for producing yeast having high glutathione content, culture thereof, fraction thereof, yeast extract, and food and drink containing glutathione - Google Patents

Description

  The present invention relates to a yeast mutant of the genus Candida. More specifically, a mutant of Candida utilis that can contain a large amount of reduced or oxidized glutathione in the bacterial body, a culture obtained by culturing the mutant, a fraction, a yeast extract, and a food and drink using these About.

  Glutathione is a tripeptide composed of three amino acids, glutamic acid, glycine, and cysteine. It is widely distributed in tissues such as the liver and muscle of yeast and animals, and is involved in detoxification and redox balance in vivo. ing. Focusing on this physiological activity, glutathione has been widely used as a therapeutic agent for liver diseases such as a therapeutic agent for alcoholic fatty liver.

  On the other hand, in recent years, as health functional foods have attracted attention from a health-oriented viewpoint, foods containing the same substance are attracting attention in the food field based on the physiological activity of glutathione.

  As an industrial production method of glutathione, a method of extracting from yeast is common, but in this case, obtaining a yeast having a high glutathione content is a necessary condition.

  As a conventionally known method for producing a glutathione-rich yeast, a method of adding an amino acid to a medium (Patent Document 1), a method of restricting zinc ions (Patent Document 2), and a method of culturing at a culture temperature lower than usual (Patent Document 2) Patent Document 3) and the like. In addition, there are ethionine / sulfite resistant strains (Patent Document 4), polyene antibiotic resistant strains (Patent Document 5), and the like by mutagen treatment.

However, in order to obtain glutathione industrially inexpensively, a further yeast having a high glutathione content is desired.
JP-A-53-94089 Japanese Patent Laid-Open No. 1-141591 JP-A-60-156379 JP 59-151894 A JP 2003-284547 A

  An object of the present invention is to provide a yeast capable of retaining a large amount of glutathione in a microbial cell and a glutathione-containing food / beverage product using the yeast under the above technical background.

  As a result of intensive studies to solve the above problems, the present inventors have found that a yeast mutant having cadmium resistance can contain glutathione in a larger amount of cells than general wild yeast. The invention has been completed.

That is, the present invention is as follows.
(1) A yeast mutant strain that is Candida utilis ABYC1560 (accession number FERMP-20094), which has cadmium resistance and can contain glutathione at a high concentration.
(2) the yeast glutathione intracellular content of the mutant strain is not less than 0.3 wt% (1) yeast mutant according.
( 3 ) A method for producing a yeast having a high glutathione content, wherein the yeast mutant according to (1) or (2) is aerobically cultured and glutathione is accumulated at a high concentration in the yeast. .
( 4 ) A culture obtained by culturing the yeast mutant according to (1) or (2) under aerobic conditions.
( 5 ) A fraction of the culture containing glutathione obtained by culturing the yeast mutant strain according to (1) or (2) under aerobic conditions.
( 6 ) A yeast extract produced using a culture obtained by culturing the mutant yeast strain according to (1) or (2) under aerobic conditions.
( 7 ) A dry yeast cell produced using a culture obtained by culturing the yeast mutant strain according to (1) or (2) under aerobic conditions.
( 8 ) A culture obtained by culturing the yeast mutant strain according to (1) or (2) under aerobic conditions, a fraction of the culture containing glutathione, or the culture or fraction subjected to heat treatment A glutathione-containing food or drink comprising a painting.

  The novel yeast mutant strain of the present invention can contain glutathione at a high concentration in the microbial cells. The novel yeast mutant strain of the present invention is aerobically cultured to have a high concentration of glutathione in the yeast microbial cells. Can be accumulated.

  Therefore, the novel yeast mutant of the present invention can be cultured under aerobic conditions to obtain a culture or fraction containing glutathione at a high concentration.

  Moreover, the novel yeast mutant of this invention can be cultured on aerobic conditions, and the yeast extract containing glutathione in high concentration can be obtained.

  Furthermore, a culture obtained by culturing the novel yeast mutant of the present invention under aerobic conditions, a fraction of the culture containing glutathione, or the culture or fraction subjected to heat treatment is contained. Thus, a glutathione-containing food or drink can be obtained.

  Hereinafter, the present invention will be described in detail.

  The novel yeast mutant of the present invention is a yeast mutant having cadmium resistance and capable of containing glutathione at a high concentration.

  In addition, the yeast mutant of the present invention can be obtained by using a Candida utilis 15-10 strain or the like as a parent strain and selecting a mutant strain having enhanced heavy metal resistance performance compared to the parent strain by mutation treatment.

  Specifically, a medium containing a heavy metal such as cadmium is subjected to a mutation treatment using a mutagen such as ultraviolet light, ionizing radiation, nitrous acid, nitrosoguanidine, ethyl methanesulfonate (hereinafter abbreviated as “EMS”). Select a mutant strain that has grown.

  The amount of glutathione contained in the cells of the selected mutant strain is measured, a mutant strain having an enhanced glutathione amount is further selected, and a mutant strain that can contain glutathione at a high concentration is searched. As the heavy metal used at this time, cadmium, mercury, or the like is used. Thus, in the present invention, a novel mutant Candida utilis ABYC1560 was obtained.

  The mutant Candida utilis ABYC1560 of the present invention accumulates 0.3 wt% or more of glutathione per dry weight in the cell. Glutathione in the present invention means the total amount of glutathione in reduced and oxidized glutathione.

  Further, the general mycological properties of the novel mutant Candida utilis ABYC1560 strain of the present invention are exactly the same as those of Candida utilis except for cadmium resistance. The novel mutant Candida utilis ABYC1560 strain of the present invention was entrusted as “Accession Number FERM P-20094”.

  In the present invention, there is further provided a method for producing a glutathione-rich yeast that aerobically cultures the yeast mutant. In order to carry out the present invention, the cadmium resistant mutant obtained by the above-described method may be aerobically cultured in a medium containing a carbon source, a nitrogen source, an inorganic salt and the like.

  As the medium composition of these strains, one or more selected from the group consisting of glucose, sucrose, acetic acid, ethanol, molasses, sulfite pulp waste liquid and the like used for culturing ordinary microorganisms as a carbon source are used. The nitrogen source is selected from the group consisting of urea, ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, and other inorganic salts, and nitrogen-containing organic substances such as corn steep liquor (CSL), casein, yeast extract, or peptone. Species or two or more are used. Furthermore, phosphoric acid component, potassium component, and magnesium component may be added to the medium. These include normal industrial products such as lime superphosphate, ammonium phosphate, potassium chloride, potassium hydroxide, magnesium sulfate, and magnesium hydrochloride. The raw material can be used. In addition, inorganic salts such as zinc, copper, manganese, and iron ions may be used. In addition, vitamins and nucleic acid-related substances may be added.

  The culture format may be batch culture, fed-batch culture, or continuous culture, but industrially fed-batch culture or continuous culture is employed.

  The culture temperature may be according to general yeast culture conditions, for example, 20 to 40 ° C., preferably 25 to 35 ° C., and the pH is preferably 3.5 to 8.0, particularly 4.0 to 6.0.

  Although the culture containing high-concentration glutathione in yeast is obtained by the method of the present invention, a fraction containing glutathione may be obtained from the culture. As a method for fractionating a fraction containing glutathione from a culture, any method may be used as long as it is a commonly performed method, and examples thereof include hot water extraction and extraction by cell disruption. Further, by loading the obtained extract on a carrier, it can be concentrated to a fraction containing glutathione at a high concentration.

  Moreover, you may prepare a yeast extract from the culture cultured by the said method. As a method for preparing a yeast extract, any method can be used as long as it is a commonly used method, but an autolysis method, an enzymatic decomposition method, an alkali extraction method, or the like is industrially employed.

  Moreover, you may prepare a dry yeast microbial cell from the culture cultured by the said method. As a method for preparing dry yeast cells, any method can be used as long as it is a usual method, but industrially, freeze-drying method, spray-drying method, drum-drying method and the like are adopted. The

  Furthermore, this invention relates to the food / beverage products containing the culture obtained by culture | cultivating the said yeast mutant under aerobic conditions, and the fraction of the said culture containing glutathione.

  These foods and drinks may be any foods and drinks that can usually be added with dry yeast and yeast extract. Examples include alcoholic beverages, soft drinks, fermented food seasonings, soups, general foods, and confectionery. be able to.

  In order to produce the food or drink of the present invention, in the process of producing the food or drink, a culture obtained by culturing the yeast mutant under aerobic conditions, or a fraction of the culture containing glutathione, is added. That's fine.

  Therefore, according to the present invention, a food or drink containing glutathione at a high concentration can be efficiently produced.

  Hereinafter, the present invention will be described with reference to examples. The present invention is not limited to these examples.

  The cell weight was measured from the weight after the culture solution was washed twice with a centrifuge and dried at 105 ° C. for 5 hours. Quantification of total glutathione combined with reduced and oxidized glutathione was measured according to the method of Titze et al. (“Analytical Biochemistry” Vol. 27, page 502, 1969). The total amount of glutathione (% (w / w)) in the dry cells was calculated by dividing the total amount of glutathione obtained by the dry cell weight, and was used as a percentage.

  Candida utilis was cultured until the logarithmic growth phase in a test tube containing YPD medium (glucose 2%, polypeptone 2%, yeast extract 1%). Then, this microbial cell was collect | recovered and the mutation process was performed using EMS in accordance with the conventional method. Mutation treatment was performed under conditions that resulted in a death rate of about 70%.

  The 15-10 strain subjected to the mutation treatment as described above was inoculated into a YPD medium containing 0.2 mg / ml of cadmium, and statically cultured at 30 ° C. for 48 hours. As a result, 200 cadmium resistant mutants were isolated. For these cadmium-resistant mutant strains, the amount of total glutathione was measured by the method described above to obtain ABYC1560 strain (accession number FERM P-20094) in which the intracellular glutathione amount was enhanced.

  The ABYC1560 strain (accession number FERM P-20094) that acquired cadmium resistance and its parent strain 15-10 were pre-cultured in YPD medium for 18 hours, then inoculated into YPD medium, and main culture was performed at 30 ° C for 18 hours. went. The cultured cells were collected by centrifugation, washed twice with water, then boiled in a boiling water bath for 5 minutes to extract glutathione in the cells, and the total amount of glutathione was measured according to the method described above. The total glutathione content in the dried cells was 0.52 wt% in the parent strain (15-10), whereas it was 1.00 wt% in the cadmium resistant strain (ABYC1560 strain (Accession No. FERM P-20094)).

  The present invention has industrial applicability in the following points.

  The novel yeast mutant strain of the present invention can contain glutathione at a high concentration in the microbial cells. The novel yeast mutant strain of the present invention is aerobically cultured to have a high concentration of glutathione in the yeast microbial cells. Can be accumulated.

  Therefore, the novel yeast mutant of the present invention can be cultured under aerobic conditions to obtain a culture or fraction containing glutathione at a high concentration.

  Moreover, the novel yeast mutant of this invention can be cultured on aerobic conditions, and the yeast extract containing glutathione in high concentration can be obtained.

  Furthermore, a culture obtained by culturing the novel yeast mutant of the present invention under aerobic conditions, a fraction of the culture containing glutathione, or the culture or fraction subjected to heat treatment is contained. Thus, a glutathione-containing food or drink can be obtained.

Claims (8)

A yeast mutant strain that is Candida utilis ABYC1560 (Accession No. FERMP-20094) that has cadmium resistance and can contain glutathione at a high concentration.   The yeast mutant according to claim 1, wherein the yeast mutant has a glutathione intracellular content of 0.3 wt% or more. A method for producing a yeast having a high glutathione content, wherein the yeast mutant according to claim 1 or 2 is cultured aerobically and glutathione is accumulated at a high concentration in the yeast. The culture obtained by culture | cultivating the yeast mutant of Claim 1 or 2 on aerobic conditions. A fraction of the culture containing glutathione obtained by culturing the yeast mutant according to claim 1 or 2 under aerobic conditions. The yeast extract manufactured using the culture obtained by culture | cultivating the yeast variant of Claim 1 or 2 on aerobic conditions. A dry yeast cell produced using a culture obtained by culturing the yeast mutant according to claim 1 or 2 under an aerobic condition. A glutathione-containing food or drink comprising a culture obtained by culturing the yeast mutant according to claim 1 or 2 under aerobic conditions, and a fraction of the culture containing glutathione.